Bag proteins and nucleic acid molecules encoding them

ABSTRACT

The present invention provides a family of BAG-1 related proteins from humans (BAG-1L, BAG-1, BAG-2, BAG-3, BAG-4 and BAG-5), the invertebrate  C. elegans  (BAG-1, BAG-2) and the fission yeast  S. pombe  (BAG-1A, BAG-1B) and the nucleic acid molecules that encode them.

This application is a divisional of U.S. Ser. No. 10/782,627, filed Feb.18, 2004, which is a continuation of U.S. Ser. No. 09/394,142, filedSep. 9, 1999, now U.S. Pat. No. 6,696,558, which claims the benefit ofU.S. Provisional Application No. 60/155,212, filed Sep. 9, 1998, whichwas converted from U.S. Ser. No. 09/150,489, each of which isincorporated herein by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under grant numberCA-67329 awarded by the National Institutes of Health. The United StatesGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the fields of molecular biology andmolecular medicine and more specifically to a novel family of proteinsthat can regulate protein folding. The functions of these proteins arepotentially diverse, including promoting tumor cell growth andmetastasis.

2. Background Information

The Hsc70/Hsp70-family of molecular chaperones participate in proteinfolding reactions, controlling protein bioactivity, degradation, complexassembly/disassembly, and translocation across membranes. These proteinsinteract with hydrophobic regions within target proteins via a carboxyl(C)-terminal peptide binding domain, with substrate binding and releasebeing controlled by the N-terminal ATP-binding domain of Hsc70/Hsp70.

Hsc70/Hsp70-assisted folding reactions are accomplished by repeatedcycles of peptide binding, refolding, and release, which are coupled toATP hydrolysis by the ATP-binding domain (ATPase) of Hsc70/Hsp70 and bysubsequent nucleotide exchange. The chaperone activity of mammalianHsc70/Hsp70 is regulated by partner proteins that either modulate thepeptide binding cycle or that target the actions of these chaperones tospecific proteins and subcellular compartments. DnaJ-family proteins(Hdj-1/Hsp40; Hdj-2; Hdj-3) stimulate the ATPase activity ofHsc70/Hsp70, resulting in the ADP-bound state which binds tightly topeptide substrates. The Hip protein collaborates with Hsc70/Hsp70 andDnaJ homologues in stimulating ATP hydrolysis, and thus also stabilizeHsc70/Hsp70 complexes with substrate polypeptides, whereas the Hopprotein may provide co-chaperone functions through interactions with theC-terminal peptide binding domain.

The Bcl-2 associated athanogene-1 (bag-1) is named from the Greek wordathanos, which refers to anti-cell death. BAG-1 was previously referredto as Bcl-2-associated protein-1 (BAP-1) in U.S. Pat. No. 5,539,094issued Jul. 23, 1996, which is incorporated herein by reference. In thisearlier patent, BAG-1 is described as a portion of the human BAG-1protein, absent the N-terminal amino acids 1 to 85. In addition, a humanprotein essentially identical to human BAG-1 was described by Zeiner andGehring, (Proc. Natl. Acad. Sci., USA 92:11465-11469 (1995)). Subsequentto the issuance of U.S. Pat. No. 5,539,094 the N-terminal amino acidsequence from 1 to 85 of human BAG-1 was reported.

BAG-1 and its longer isoforms BAG-1M (Rap46) and BAG-1L are recentlydescribed Hsc70/Hsp70-regulating proteins. BAG-1 competes with Hip forbinding to the Hsc70/Hsp70 ATPase domain and promotes substrate release.BAG-1 also reportedly stimulates Hsc70-mediated ATP hydrolysis byaccelerating ADP/ATP exchange, analogous to the prokaryotic GrpEnucleotide exchange protein of the bacterial Hsc70 homologue, DnaK. Genetransfection studies indicate that BAG-1 proteins can influence a widevariety of cellular phenotypes through their interactions withHsc70/Hsp70, including increasing resistance to apoptosis, promotingcell proliferation, enhancing tumor cell migration and metastasis, andaltering transcriptional activity of steroid hormone receptors.

Despite the notable progress in the art, there remains an unmet need forthe further identification and isolation of additional homologous BAGprotein species, and the nucleic acid molecules and/or nucleotidesequences that encode them. Such species would provide additional meansby which the identity and composition of the BAG domain, that is, theportion of the protein that is influencing or modulating proteinfolding, could be identified. In addition, such species would be usefulfor identifying agents that modulate apoptosis as candidates fortherapeutic agents, in particular, anticancer agents. The presentinvention satisfies these need, as well as providing substantial relatedadvantages.

SUMMARY OF THE INVENTION

The present invention provides a family of BAG-1 related proteins fromhumans [BAG-1L (SEQ ID NO:2), BAG-1 (beginning at residue 116 of SEQ IDNO:2), BAG-2 (SEQ ID NO: 4), BAG-3 (SEQ ID NO:6) and (SEQ ID NO:20),BAG-4 (SEQ ID NO:8) and (SEQ ID NO:22) and BAG-5 (SEQ ID NO:10) and (SEQID NO:24)], the invertebrate C. elegans [BAG-1 (SEQ ID NO:12), BAG-2(SEQ ID NO: 14)] and the fission yeast S. pombe [BAG-1A (SEQ ID NO:16),BAG-1B (SEQ ID NO:18)] and the nucleic acid molecules that encode them.

Another aspect of the present invention provides an amino acid sequencepresent in the family of BAG-1 related proteins, that modulatesHsc70/Hsp70 chaperone activity, that is, the BAG domain.

Another aspect of the present invention provides novel polypeptide andnucleic acid compositions and methods useful in modulating Hsc70/Hsp70chaperone activity.

Another aspect of the present invention is directed to methods fordetecting agents that modulate the binding of the BAG family ofproteins, such as BAG-1 (beginning at residue 116 of SEQ ID NO:2), andrelated proteins with the Hsc70/Hsp70 Family of proteins or with otherproteins that may interact with the BAG-Family proteins.

Still another aspect of the present invention is directed to methods fordetecting agents that induce the dissociation of a bound complex formedby the association of BAG-Family proteins with Hsc70/Hsp70 Familymolecule chaperones or other proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the full length cDNA sequence for human BAG-1 (SEQ ID NO:1)protein with the corresponding amino acid sequence (SEQ ID NO:2). Withinthe full length sequence are included the overlapping sub-sequences ofBAG-1 (beginning at nucleotide 391), BAG-1M [beginning at nucleotide 260of (SEQ ID NO:2)], and BAG-1L [beginning at nucleotide 46 of (SEQ IDNO:2)].

FIGS. 2A and 2B combined shows the full length cDNA sequence (SEQ IDNO:3) aligned with the corresponding amino acid residues for human BAG-2protein (SEQ ID NO:4).

FIG. 3 shows a cDNA sequence (SEQ ID NO:5) aligned with thecorresponding amino acid residues for human BAG-3 protein (SEQ ID NO:6).

FIG. 4 shows the a cDNA sequence (SEQ ID NO:7) aligned with thecorresponding amino acid residues for human BAG-4 protein (SEQ ID NO:8).

FIG. 5 shows a cDNA sequence (SEQ ID NO:9) aligned with thecorresponding amino acid residues for human BAG-5 protein (SEQ ID NO:10).

FIG. 6A shows the full length cDNA sequence for C. elegans BAG-1 protein(SEQ ID NO:11).

FIG. 6B shows the 210 amino acid sequence for C. elegans BAG-1 protein(SEQ ID NO:12).

FIG. 7A shows the full length cDNA sequence for C. elegans BAG-2 protein(SEQ ID NO:13).

FIG. 7B shows the 458 amino acid sequence for C. elegans BAG-2 protein(SEQ ID NO:14).

FIG. 8A shows the full length cDNA sequence for S. pombe BAG-1A protein(SEQ ID NO:15).

FIG. 8B shows the 195 amino acid sequence for S. pombe BAG-1A protein(SEQ ID NO:16).

FIG. 9A shows the full length cDNA sequence for S. pombe BAG-1B protein(SEQ ID NO:17).

FIG. 9B shows the 206 amino acid sequence for S. pombe BAG-1B protein(SEQ ID NO:18).

FIG. 10 shows the topologies of the BAG-family proteins; human BAGproteins, BAG-1 (SEQ ID NO:2), BAG-2 (SEQ ID NO:4), BAG-3 (SEQ ID NO:6),BAG-4 (SEQ ID NO:8), BAG-5 (SEQ ID NO:10); S. pombe BAG-1A (SEQ IDNO:16) and BAG-1B (SEQ ID NO:18); and C. elegans BAG-1 (SEQ ID NO:12)and BAG-2 (SEQ ID NO:14). (A) The relative positions of the BAG domainsare shown in black, ubiquitin-like regions are represented in gray, WWdomain are represented in strips. Nucleoplasmin-like nuclearlocalization sequence are also shown. (B) The amino acid sequences ofthe BAG domain for human BAG-1 (SEQ ID NO:2), BAG-2 (SEQ ID NO:4), BAG-3(SEQ ID NO:6), BAG-4 (SEQ ID NO:8), BAG-5 (SEQ ID NO:10), S. pombeBAG-1A (SEQ ID NO:16) and BAG-1B (SEQ ID NO:18), and C. elegans BAG-1(SEQ ID NO:12) and BAG-2 (SEQ ID NO:14) are aligned demonstrating theirhomology. Black and gray shading represent identical and similar aminoacids, respectively.

FIG. 11 shows assays demonstrating the interaction of BAG-familyproteins with Hsc70/ATPase. (A) Two-hybrid assays using yeast expressingthe indicated fusion proteins. Blue color indicates a positiveinteraction, resulting in activation of the lacZ reporter gene. (B) Invitro protein assays using GST-fusion proteins and ³⁵S-labeled in vitrotranslated proteins. (C) Co-immunoprecipitation assays using anti-FLAG™or IgG1 control antibodies and lysates from 293T cells expressingFLAG™-tagged BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQID NO:4), BAG-3 (SEQ ID NO:6), Daxx, or Apaf-1.

FIG. 12 shows surface plasmon resonance analysis of BAG-family proteininteractions with Hsc70/ATPase. (A) SDS-PAGE analysis of purifiedrecombinant proteins. (B) Representative SPR results of biosensor chipscontaining immobilized BAG proteins with and without maximally boundHsc70/ATPase.

FIG. 13 shows representative SPR results for biosensor chips containingimmobilized BAG-1 (beginning at residue 116 at SEQ ID NO:2), BAG-1 (ΔC),BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6) proteins. Hsc70/ATPase wasflowed over the chips (arrow/left) until maximal binding was reached(response units), then flow was continued without Hsc70/ATPase(arrow/right). For BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6), Hsc70was injected at 0.0175, 0.035, 0.07, 0.14, and 0.28 μM.

FIG. 14 shows BAG-family protein modulation of Hsc70 chaperone activity.(A) Protein refolding assay of chemically-denatured luciferase by Hsc70plus DnaJ in the absence or presence of BAG and BAG-mutant proteins. (B)Concentration-dependent inhibition of Hsc70-mediated protein refoldingby BAG-family proteins [BAG-1 (beginning at residue 116 of SEQ ID NO:2),BAG-2 (SEQ ID NO:4), BAG-3 (SEQ ID NO:6)] but not by BAG-mutant (BAG-1(ΔC). (C) Hsc70/Hsp40-mediated refolding of heat-denatured luciferasewas assayed in the presence of (black bars) or absence of (striped bars)of 1.8 μM Hip, with (lanes 3-10) or without (lanes 1,2) variousBAG-family proteins (1.8 μM) as indicated (mean±SE; n=3). A control(CNTL) is shown (lane 1) in which Hsc70 was replaced with an equivalentamount of BSA.

FIGS. 15A and 15B show an expanded cDNA sequence for human BAG-3 protein(SEQ ID NO:19).

FIG. 15C shows the corresponding amino acid residues for the human BAG-3protein (SEQ ID NO:20) of FIGS. 15A and 15B.

FIGS. 15D and 15E show the expanded cDNA sequence (SEQ ID NO: 19)aligned with the corresponding amino acid residues for human BAG-3protein of FIGS. 15A and 15B (SEQ ID NO:20).

FIGS. 16A and 16B show an expanded cDNA sequence for human BAG-4 protein(SEQ ID NO:21).

FIG. 16C shows the corresponding amino acid residues for the human BAG-4protein of FIGS. 16A and 16B (SEQ ID NO:22).

FIGS. 16D and 16E show the expanded cDNA sequence (SEQ ID NO:21) alignedwith the corresponding amino acid residues for human BAG-4 protein ofFIGS. 16A and 16B (SEQ ID NO:22).

FIGS. 17A, 17B, 17C, and 17D show an expanded cDNA sequence for humanBAG-5 protein (SEQ ID NO:23).

FIG. 17E shows the corresponding amino acid residues for the human BAG-5protein of FIG. 17A-17D (SEQ ID NO:24).

FIGS. 17F, 17G and 17H show the expanded cDNA sequence (SEQ ID NO:23)aligned with the corresponding amino acid residues for human BAG-5protein of FIG. 17A-17D (SEQ ID NO:24).

FIG. 18 shows the topologies of the BAG-family proteins; human BAGproteins, BAG-1 (SEQ ID NO:2), BAG-2 (SEQ ID NO:4), expanded BAG-3 (SEQID NO:20), expanded BAG-4 (SEQ ID NO:22), expanded BAG-5 (SEQ ID NO:24);S. pombe BAG-1A (SEQ ID NO:16) and BAG-1B (SEQ ID NO:18); and C. elegansBAG-1 (SEQ ID NO:12) and BAG-2 (SEQ ID NO: 14). The relative positionsof the BAG domains are shown in black, ubiquitin-like regions arerepresented in gray, WW domain are represented in strips.Nucleoplasmin-like nuclear localization sequence are also shown.

DEFINITIONS

The term “apoptosis”, as used herein, refers to the process ofprogrammed cell death, although not all programmed cell deaths occurthrough apoptosis, as used herein, “apoptosis” and “programmed celldeath” are used interchangeably.

The term “tumor cell proliferation”, as used herein refers to theability of tumor cells to grow and thus expand a tumor mass.

The term “cell migration”, as used herein refers to the role cellmotility plays in the invasion and potentially metastasis by tumorcells.

The term “metastasis”, as used herein refers to the spread of a diseaseprocess from one part of the body to another, as in the appearance ofneoplasms in parts of the body remote from the site of the primarytumor; results in dissemination of tumor cells by the lymphatics orblood vessels or by direct extension through serious cavities orsubarachnoid or other spaces.

The term “steroid hormone receptor function”, as used herein refers tophysiological, cellular and molecular functioning of receptors sitesthat bind with steroid hormones.

The term “substantially purified”, as used herein, refers to nucleicacid or amino acid sequence that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

“Nucleic acid molecule” as used herein refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single or doublestranded, and represent the sense or antisense strand.

“Hybridization”, as used herein, refers to any process by which a strandof nucleic acid binds with a complementary strand through base pairing.

The terms “complementary” or “complementarity”, as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence “A-G-Tbinds to the complementary sequence “T-C-A”.

The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid and is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency.

The term “antisense”, as used herein, refers to nucleotide sequenceswhich are complementary to a specific DNA or RNA sequence. The term“antisense strand” is used in reference to a nucleic acid strand that iscomplementary to the “sense” strand. Antisense molecules may be producedby any method, including synthesis by ligating the gene(s) of interestin a reverse orientation to a viral promoter which permits the synthesisof a complementary strand. Once introduced into a cell, this transcribedstrand combines with natural sequences produced by the cell to formduplexes. These duplexes then block either the further transcription ortranslation. In this manner, mutant phenotypes may be generated. Thedesignation “negative” is sometimes used in reference to the antisense,and “positive” is sometimes used in reference to the sense strand.

“Amino acid sequence” as used herein refers to an oligopeptide, peptide,polypeptide, or protein sequence, and fragments or portions thereof, andto naturally occurring or synthetic molecules. Where “amino acidsequence” is recited herein this term excludes an amino acid sequence ofa naturally occurring protein. “Amino acid sequence”, “polypeptide” or“protein” are not meant to limit the amino acid sequence to thecomplete, native amino acid sequence associated with the recited proteinmolecule.

The term “functional fragments” or “fragments”, as used herein, withregard to a protein refers to portions of that protein that are capableof exhibiting or carrying out the activity exhibited by the protein as awhole. The portions may range in size from three amino acid residues tothe entire amino acid sequence minus one amino acid. For example, aprotein “comprising at least a functional fragment of the amino acidsequence of SEQ ID NO:1”, encompasses the full-length of the protein ofSEQ ID NO:1 and portions thereof.

A “derivative” of a BAG protein, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The derivative mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., substitution of anapolar amino acid with another apolar amino acid (such as replacement ofleucine with isoleucine). The derivative may also have “nonconservative”changes, wherein a substituted amino acid has different but sufficientlysimilar structural or chemical properties that permits such asubstitution without adversely effecting the desired biologicalactivity, e.g., replacement of an amino acid with an uncharged polar Rgroup with an amino acid with an apolar R group (such as replacement ofglycine with tryptophan), or alternatively replacement of an amino acidwith a charged R group with an amino acid with an uncharged Polar Rgroup (such as replacement of lysine with asparagine).

Amino Acids - Apolar R Groups Abbreviations Amino Acid Radical 3-Letter1-Letter alanine methyl ala A valine 2-propyl aal V leucine2-methylpropyl leu L isoleucine 2-butyl ile I proline propyl* - cyclizedpro P phenylalanine benzyl phe F trytophan 3-indolylmethl tyr Wmethionine methylthioethyl met M

Amino Acids - Uncharged Polar R Groups Abbreviations Amino Acid Radical3-Letter 1-Letter glycine H gly G serine hydroxymethyl ser S threonine1-hydroxyethyl thr T cysteine thiolmethyl cys C tyrosine4-hydroxyphenylmethyl tyr Y asparagine aminocarbonylmethyl asn Nglutamine aminocarbonylethyl gln Q

Amino Acids - Charged R Groups Abbreviations Amino Acid Radical 3-Letter1-Letter aspartic acid carboxymethyl asp D glutamic acid carboxyethylglu E lysine 4-aminobutyl lys K arginine 3-guanylpropyl arg R histidine4-imidazoylmethyl his H

Similar minor modifications may also include amino acids deletions orinsertions or both. Guidance in determining which amino acid residuesmay be modified as indicated above without abolishing the desiredbiological functionality may be determined using computer programs wellknown in the art, for example, DNASTAR software. In addition, thederivative may also result from chemical modifications to the encodedpolypeptide, including but not limited to the following, replacement ofhydrogen by an alkyl, acyl, or amino group; esterification of a carboxylgroup with a suitable alkyl or aryl moiety; alkylation of a hydroxylgroup to form an ether derivative. Further a derivative may also resultfrom the substitution of a L-configuration amino acid with itscorresponding D-configuration counterpart.

The term “mimetic”, as used herein, refers to a molecule, the structureof which is developed from knowledge of the structure of aprotein/polypeptide or portions thereof (such as BAG-1) and, as such, isable to effect some or all of the actions of BAG-1 protein.

“Peptide nucleic acid”, as used herein, refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al.,Anticancer Drug Des. 8:53-63 (1993)).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a family of BAG-1 related proteins fromhumans [BAG-1L (SEQ ID NO:2), BAG-1S beginning at residue 116 of SEQ IDNO:2, BAG-2 (SEQ ID NO:4), BAG-3 (SEQ ID NO:6) and (SEQ ID NO:20), BAG-4(SEQ ID NO: 8) and (SEQ ID NO:22) and BAG-5 (SEQ ID NO:10) and (SEQ IDNO:24)], the invertebrate C. elegans [BAG-1 (SEQ ID NO:12), BAG-2 (SEQID NO: 14)] and the fission yeast S. pombe [BAG-1A (SEQ ID NO:16),BAG-1B (SEQ ID NO:18)], specifically the full length amino acidsequences comprising human BAG-1L (SEQ ID NO:2), BAG-1 (beginning atresidue 116 of SEQ ID NO:2), and BAG-2 (SEQ ID NO:4) C. elegans BAG-1(SEQ ID NO:12), and BAG-2 (SEQ ID NO:14), and S. pombe BAG-1A (SEQ IDNO:16) and BAG-1B (SEQ ID NO:18); and partial sequences comprising humanBAG-3 (SEQ ID NO: 6) and (SEQ ID NO:20), BAG-4 (SEQ ID NO:8) and (SEQ IDNO:22), and BAG-5 (SEQ ID NO:10) and (SEQ ID NO:24) and functionalfragments thereof. In particular, the invention provides the amino acidsequences comprising human BAG-2 (SEQ ID NO:4), BAG-3 (SEQ ID NO:6) and(SEQ ID NO:20), BAG-4 (SEQ ID NO:8) and (SEQ ID NO:22), and BAG-5 (SEQID NO:10) and (SEQ ID NO:24) proteins.

Another aspect of the present invention provides the nucleic moleculeand nucleotide sequences that encode the family of BAG-1 relatedproteins from humans [BAG-1 (SEQ ID NO:1), BAG-2 (SEQ ID NO:3), BAG-3(SEQ ID NO:5) and (SEQ ID NO:19), BAG-4 (SEQ ID NO:7) and (SEQ ID NO:21)and BAG-5 (SEQ ID NO:9) and (SEQ ID NO:23)], the invertebrate C. elegans[BAG-1 (SEQ ID NO:11), BAG-2 (SEQ ID NO:13)] and the fission yeast S.pombe [BAG-1A (SEQ ID NO:15), BAG-1B (SEQ ID NO:17)].

BAG-1L (SEQ ID NO:2) is a multifunctional protein that blocks apoptosis,promotes tumor cell metastasis, and contributes to factor-independentand p53-resistant cell growth. BAG-1L (SEQ ID NO:2) interacts withseveral types of proteins, including Bcl-2, some tyrosine kinase growthfactor receptors, steroid hormone receptors, and the p53-induced cellcycle regulator Siah-1A.

BAG-1 is a regulator of Hsc70/Hsp70 family molecular chaperones. Acarboxyl-terminal domain in this protein binds tightly to the ATPasedomains of Hsc70 and Hsp70 (K_(D)=1 nM) (Zeiner, M., Gebauer, M., andGehring, U., EMBO J. 16: 5483-5490, (1997)). BAG-1 modulates theactivity of these molecular chaperones, acting as an apparent functionalantagonist of the Hsp70/Hsc70-associated protein Hip (3-5)(Höhfeld, J.and Jentsch, S., EMBO J. 16: 6209-6216, (1997); Takayama, S., Bimston,D. N., Matsuzawa, S., Freeman, B. C., Aime-Sempe, C., Xie, Z., Morimoto,R. J., and Reed, J. C., EMBO J. 16: 4887-96, (1997); Zeiner, M.,Gebauer, M., and Gehring, U., EMBO J. 16: 5483-5490, (1997)). Ingeneral, protein refolding is accomplished by Hsp70/Hsc70 throughrepeated cycles of target peptide binding and release, coupled to ATPhydrolysis (Ellis, R., Curr Biol. 7: R531-R533, (1997)). BAG-1 appearsto promote substrate release, whereas Hip stabilizes Hsp70/Hsc70 complexformation with target peptides (Höhfeld, J., Minami, Y, and Hartl,F.-U., Cell. 83:589-598, (1995)). Since each substrate interaction withHsc70/Hsp70 is unique in terms of the optimal length of time the proteintarget should remain complexed with Hsc70/Hsp70 for achieving newconformations, the net effect of BAG-1 can be either enhancement orinhibition of the refolding reaction.

The 70 kd heat shock family proteins (Hsp70/Hsc70) are essential to avariety of cellular processes and have been implicated in cancer, yet itis unclear how these proteins are regulated in vivo. A variety ofco-chaperones have been identified which may target Hsp70/Hsc70 todifferent subcellular compartments or promote their interactions withspecific protein or protein complexes. BAG-1 appears to represent anovel Hsp70/Hsc70 regulator which differs functionally from all othermammalian co-chaperones identified to date, such as members of theDnaJ-, Hip-, Hop-, and cyclophilin-families of proteins.

Another aspect of the present invention provides the amino acid sequenceof a binding domain of about 40 to 55 amino acids that bind the aHsc70/Hsp70 ATPase domain. The BAG domain is situated near theC-terminus, and the ubiquitin-like domains are situated near theN-terminus.

The BAG family of proteins of the present invention contain a commonconserved C-terminal domain (the “BAG” domain) that facilitates bindingto the ATPase domain of Hsp70/Hsc70. The carboxyl-terminal domain ofBAG-1 binds to the ATPase domain of Hsc70/Hsp70 and regulates itschaperone function by acting as a ADP-ATP exchange factor. Other domainsof BAG-1 mediate interactions with proteins such as Bcl-2 and retinoicacid receptors (RARs), allowing BAG-1 to target Hsc70/Hsp70 to otherproteins, presumably modulating their function by changing theirconformations.

Human BAG-1 was previously shown to inhibit Hsc70/Hsp70 dependentrefolding of denatured protein substrates in vitro (S. Takayama, et al.,EMBO J. 16, 4887-96 (1997); M. Zeiner, M. Gebauer, U. Gehring, EMBO J.16, 5483-5490 (1997); and J. Höhfeld, S. Jentsch, EMBO J. 16, 6209-6216(1997)). In Example III, Part A the effects of recombinant human BAG-1,BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) were compared using in vitroprotein refolding assays similar to those employed previously forassessing BAG-1. The study showed that addition of equimolar amounts ofeach of the recombinant proteins to Hsc70 resulted in significantinhibition of luciferase refolding, with BAG-2 (SEQ ID NO:4) and BAG-3(SEQ ID NO:6) showing somewhat greater inhibitor activity than BAG-1(FIG. 4A). In a separate luciferase folding study BAG-1, BAG-2 (SEQ IDNO:4) and BAG-3 (SEQ ID NO:6) once again displayed inhibition ofluciferase refolding, however in this study varying amounts of BAG-1,BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) were added relative to Hsc70which resulting in concentration-dependent inhibition of Hsc70 chaperoneactivity, i.e., luciferase folding (Example III Part A).

Additional follow on studies using the same experimental protocols asthe previous studies, as taught in Example IIA, have shown that BAG-4(SEQ ID NO:22) also undergoes association with Hsc70/ATPase.

Yet another aspect of the present invention provides a nucleotidesequence having at least about 15 nucleotides and, generally, about 25nucleotides, preferably about 35 nucleotides, more preferably about 45nucleotides, and most preferably about 55 nucleotides that can hybridizeor is complementary under relatively stringent conditions to a portionof the nucleic acid sequences shown in FIGS. 1-9 and FIGS. 15-17, inparticular the BAG domain as shown in FIG. 1B, e.g., nucleotides 552-593of human BAG-3, or nucleotides 167-221 of human BAG-4.

Yet another aspect of the present invention provides a compound of theformula,R^(N)-R¹X¹R²X²R³X³R⁴X⁴R⁵X⁵R⁶X⁶R⁷X⁷X⁸R⁹X⁹R¹⁰X¹⁰R¹¹X¹¹-R^(C)wherein,

R^(N) is a group of 1 to 552 independently selected amino acids;

R¹ is a group of 3 independently selected amino acids;

X¹ is an amino acid with a charged or uncharged R group, such asaspartic acid, glutamic acid, asparagine, or glutamine;

R² is a group of 7 independently selected amino acids;

X² is an amino acid with a charged R group, such as glutamic acid;

R³ is a group of 5 independently selected amino acids;

X³ is an amino acid with an apolar R group, such as leucine, methionine,or isoleucine;

R⁴ is a group of 3 independently selected amino acids;

X⁴ is an amino acid with charged R group, such as aspartic acid orglutamine acid;

R⁵ is a single independently selected amino acid;

X⁵ is an amino acid with apolar or uncharged R group, such as leucine,valine, methionine, alanine or threonine;

R⁶ is a group of 15 independently selected amino acids;

-   -   X⁶ is an amino acid with a charged or uncharged R group, such as        arginine, lysine, glutamine or aspartic acid;

R⁷ is a group of 2 independently selected amino acids;

X⁷ is an amino acid with a charged R group, such as arginine;

X⁸ is an amino acid with a charged R group, such as arginine or lysine;

R⁹ is a group of 2 independently selected amino acids;

X⁹ is an amino acid with an apolar R group, such as valine;

R¹⁰ is a group of 3 independently selected amino acids;

X¹⁰ is an amino acid with an uncharged R group, such as glutamine;

R¹¹ is a group of 2 independently selected amino acids;

X¹¹ is an amino acid with an apolar R group, such as leucine; and

R^(C) is a group of 1 to 100 independently selected amino acids.

A nucleotide sequence of at least about 15 nucleotides and, generally,about 25 nucleotides, preferably about 35 nucleotides, more preferablyabout 45 nucleotides, and most preferably about 55 nucleotides can beuseful, for example, as a primer for the polymerase chain reaction (PCR)or other similar reaction mediated by a polymerase such as a DNA or RNApolymerase (see PCR Protocols: A guide to methods and applications, ed.Innis et al. (Academic Press, Inc., 1990), which is incorporated hereinby reference; see, for example, pages 40-41). In addition, such anucleotide sequence of the invention can be useful as a probe in ahybridization reaction such as Southern or northern blot analysis or ina binding assay such as a gel shift assay.

A nucleotide sequence of the invention can be particularly useful as anantisense molecule, which can be DNA or RNA and can be targeted to allor a portion of the 5′-untranslated region or of the 5′-translatedregion of a bag-1 nucleic acid sequence in a cell. For example, anantisense molecule can be directed to at least a portion of the sequenceshown as the BAG domain in FIG. 1A, e.g., nucleotides 272-319 of humanBAG-1L (SEQ ID NO:1), or nucleotides 79-147 of human BAG-5 (SEQ IDNO:9). Since the 5′-region of a nucleic acid contains elements involvedin the control of expression of an encoded protein, an antisensemolecule directed to the 5′-region of a nucleic acid molecule can affectthe levels of protein expressed in a cell.

A nucleotide sequence of the invention also can be useful as a probe toidentify a genetic defect due a mutation of a gene encoding a BAGprotein in a cell. Such a genetic defect can lead to aberrant expressionof a BAG protein in the cell or to expression of an aberrant BAGprotein, which does not properly associate with a Bcl-2-related proteinor Hsc70/Hsp70 protein in the cell. As a result, a genetic defect in agene encoding, for example, human BAG-1 can result in a pathologycharacterized by increased or decreased levels in protein folding.

Further a nucleotide compound or composition as taught in the presentinvention can be synthesized using routine methods or can be purchasedfrom a commercial source. In addition, a population of such nucleotidesequences can be prepared by restriction endonuclease or mild DNAsedigestion of a nucleic acid molecule that contains nucleotides as shownin the nucleotide sequences shown in FIGS. 1-9 and FIGS. 15-17 thatencodes the amino acids sequences also shown in FIGS. 1-9 and FIGS.15-17. Methods for preparing and using such nucleotide sequences, forexample, as hybridization probes to screen a library for homologousnucleic acid molecules are well known in the art (see, for example,Sambrook et al., Molecular Cloning: A laboratory manual (Cold SpringHarbor Laboratory Press 1989); Ausubel et al., Current Protocols inMolecular Biology (Green Publ., NY 1989), each of which is incorporatedherein by reference).

A particular nucleotide sequence can be designed based, for example, ona comparison of the nucleic acid molecules encoding any one of the BAGfamily proteins, as shown in FIGS. 1-9 and FIGS. 15-17, with another inthe family. Such a comparison allows, for example, the preparation of anucleotide sequence that will hybridize to a conserved region present inboth nucleic acid molecules, thus providing a means to identifyhomologous nucleic acid molecules present in other cell types or otherorganisms. In addition, such a comparison allows the preparation of anucleotide sequence that will hybridize to a unique region of any of theBAG family nucleotide sequences, such as those corresponding to the BAGdomain, thus allowing identification of other proteins sharing thismotif. In this regard, it is recognized that, while the human BAG-3proteins shown as FIGS. 3 and 20, and human BAG-5 proteins shown asFIGS. 5 and 24, are only partial sequences, a variant human BAG-3 orBAG-5 produced, for example, by alternative splicing can exist and canbe identified using an appropriately designed nucleotide sequence of theinvention as a probe. Such useful probes readily can be identified byinspection of the sequences shown in the disclosed Figures by acomparison of the encoding nucleotide sequences.

If desired, a nucleotide sequence of the invention can incorporate adetectable moiety such as a radiolabel, a fluorochrome, a ferromagneticsubstance, a luminescent tag or a detectable binding agent such asbiotin. These and other detectable moieties and methods of incorporatingsuch moieties into a nucleotide sequence are well known in the art andare commercially available. A population of labeled nucleotide sequencescan be prepared, for example, by nick translation of a nucleic acidmolecule of the invention (Sambrook et al., supra, 1989; Ausubel et al.,supra, 1989).

One skilled in the art would know that a method involving hybridizationof a nucleotide sequence of the invention can require that hybridizationbe performed under relatively stringent conditions such that nonspecificbackground hybridization is minimized. Such hybridization conditions canbe determined empirically or can be estimated based, for example, on therelative GC content of a sequence and the number of mismatches, ifknown, between the probe and the target sequence (see, for example,Sambrook et al., supra, 1989).

The invention further provides antibodies specific for human BAG familyprotein. As used herein, the term “antibody” includes polyclonal andmonoclonal antibodies, as well as polypeptide fragments of antibodiesthat retain a specific binding activity for human BAG-1 of at leastabout 1×10⁵ M⁻¹. One skilled in the art would know that anti-BAG-1antibody fragments such as Fab, F(ab′)₂ and Fv fragments can retainspecific binding activity for human BAG-1 (beginning at residue 116 ofSEQ ID NO:2) and, thus, are included within the definition of anantibody. In addition, the term “antibody” as used herein includesnaturally occurring antibodies as well as non-naturally occurringantibodies and fragments that retain binding activity such as chimericantibodies or humanized antibodies. Such non-naturally occurringantibodies can be constructed using solid phase peptide synthesis, canbe produced recombinantly or can be obtained, for example, by screeningcombinatorial libraries consisting of variable heavy chains and variablelight chains as described by Huse et al., Science 246:1275-1281 (1989),which is incorporated herein by reference.

One skilled in the art would know that purified BAG family protein,which can be prepared from natural sources or synthesized chemically orproduced recombinantly, or portions of a BAG family protein, including aportion of human BAG family protein such as a synthetic peptide asdescribed above, can be used as an immunogen. Such peptides useful forraising an antibody include, for example, peptide portions of theN-terminal 85 amino acids or the BAG domain of any of the human BAGproteins (see FIG. 1B). A particularly advantageous use of such aprotein is for the immunostaining, wherein the methods provides aprocess to contrast the immunostaining of BAG-family proteins incarcinoma cells with adjacent non-neoplastic prostatic epithelial andbasal cells which are generally present in the same tissue sections.These results would be correlated with a Gleason grade to determinewhether any of the BAG-family proteins tend to be expressed at higher orlower levels in histologically advanced tumors. From this process adetermination can be made as to degree at which the disease isprogressing in a given patient, i.e., a prognosis can be made.

Non-immunogenic fragments or synthetic peptides of BAG proteins can bemade immunogenic by coupling the hapten to a carrier molecule suchbovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), asdescribed in Example IV, below. In addition, various other carriermolecules and methods for coupling a hapten to a carrier molecule arewell known in the art and described, for example, by Harlow and Lane,Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press,1988), which is incorporated herein by reference.

EXAMPLES

The following examples are given to enable those skilled in the art tomore clearly understand and to practice the present invention. Theyshould not be considered as limiting the scope of the invention, butmerely as being illustrative and representative thereof.

Example I Isolation and Characterization of BAG-family cDNA Sequences

This example describes methods for isolating and characterizing ofBAG-family cDNA sequences from human, nematode and yeast.

A. Cloning of Human BAG cDNA Sequences

Yeast two-hybrid library screening of a human Jurkat cell cDNA librarywas performed as described by Takayama et al., EMBO J., 16:4887-96(1997); Matsuzawa et al., EMBO J., 17:2736-2747 (1998), which areincorporated herein by reference) using EGY48 strain yeast transformedwith pGilda-Hsc70/ATPase (67-377 amino acids) and the lacZ reporterplasmid pSH18-34. Of the resulting ^(˜)5×10⁶ transformants, 112 Leu⁺colonies were obtained after 1 week incubation at 30° C. Assay ofβ-galactosidase (β-gal) activity of these colonies resulted in 96clones. Mating tests were then performed using RFY206 yeast straintransformed with pGilda, pGilda mBAG-1 (1-219), or pGilda Hsc70/ATPase.Of these, 66 displayed specific interactions with Hsc70/ATPase. ThepJG4-5 cDNAs were recovered using KC8 E. coli strain which isauxotrophic for tryptophan (Trp). DNA sequencing revealed 3 partiallyoverlapping human BAG-1, 4 identical and one overlapping cDNAs encodingBAG-2, and 2 partially overlapping BAG-3 clones.

Using the above described yeast two-hybrid screen with the ATPase domainof Hsc70 as “bait”, several human cDNAs were cloned which encodeportions of BAG-1 or of two other BAG-1-like proteins which are termedBAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6). The longest of the cDNAsfor BAG-2 (SEQ ID NO:3) and BAG-3 (SEQ ID NO:5) contained open readingframes (ORFs) of 207 and 162 amino acids, respectively, followed by stopcodons. All BAG-1 (SEQ ID NO:1), BAG-2 (SEQ ID NO:3) and BAG-3 (SEQ IDNO:5) cDNAs obtained by two-hybrid library screening with Hsc70/ATPasecontained a conserved domain of about 40-50 amino acids which are termedthe “BAG” domain and are shown in FIG. 10. These results demonstratethat a family of BAG-1-related proteins all contain a conserved ˜45amino acid region near their C-terminus that binds Hsc70/Hsp70.

B. Identification of Additional BAG-family Proteins

A search of the translated Genbank database using the bBLAST and FASTAsearch programs also identified human ESTs that provided sequences forfurther investigation of BAG-family proteins. The putative BAG-4 (SEQ IDNO:8) and BAG-5 (SEQ ID NO:10) proteins contain BAG-domains that sharethe greatest sequence similarity with the BAG-domain of BAG-3 (SEQ IDNO:6). These were designated BAG-4 (Accession number AA693697, N74588)and BAG-5 (Accession number AA456862, N34101). BAG-4 has 62% identityand ˜81% similarity to BAG-3, and BAG-5 has 51% identity and ˜75%similarity to BAG-3.

Additional BAG-family orthologues or homologues were also identifiedusing computer-based searches and resulted in BAG-family homologue inthe nematode C. elegans and the fission yeast S. pombe. The C. elegansgenome encodes two apparent BAG-family proteins, which are most similarin their overall sequences to the human BAG-1 (Afo39713, gi:2773211)(SEQ ID NO:12) and BAG-2 (SEQ ID NO:14) (Afo68719, gi:3168927). The S.pombe contains two BAG-family proteins that share the greatest overallsequence similarity with human BAG-1 (Alo23S54, gi/3133105 and Alo23634,gi/3150250). The human and C. elegans BAG-1 proteins as well as S. pombeBAG-1A all have ubiquitin-like domains near their N-termini (see FIG.10A) of unknown function.

The overall predicted amino acid sequences of the C. elegans BAG-1 (SEQID NO:12) and S. pombe BAG-1A (SEQ ID NO:16) proteins are ˜18% identical(^(˜)61% similar) and ^(˜)17% identical (^(˜)64% similar), respectively,to human BAG-1, implying origin from a common ancestral gene. The C.elegans BAG-1 protein (SEQ ID NO:12), however, contains a 5 to 7 aminoacid insert in its BAG-domain as compared to the human, murine, andyeast BAG-1 homologues (see FIG. 10B), and is more similar to BAG-2 (SEQID NO:4) in regard to its BAG-domain. C. elegans and human BAG-2 alsomay be derived from a common ancestor as the C-terminal 225 amino acidregion which encompasses both the BAG domain and upstream region of bothC. elegans and human BAG-2 share ^(˜)34% amino acid sequence identityand ^(˜)70% similarity. The human BAG-2 protein (SEQ ID NO:4), however,contains a 9 amino acid insert in its BAG-domain compared to it C.elegans counterpart (see FIG. 10B). Evolutionary-tree predictionalgorithms suggest that human and C. elegans BAG-2 represent a distinctbranch of the BAG-family that is more evolutionarily distant from theother BAG-family proteins. None of the predicted BAG-family proteinscontain recognizable regions analogous to those found in other Hsc70regulatory proteins, such as the J-domains and G/F-domains of DnaJfamily proteins and the Tetratricopeptide Repeat (TR) domains of Hip/Hopfamily proteins.

C. Yeast Two-hybrid Assay of BAG Binding to Hsc70/ATPase

The longest of the cDNAs obtained for the BAG-2 and BAG-3 proteins wereexpressed with N-terminal transactivation (TA) domains in yeast andtested by yeast two-hybrid assay for interactions with fusion proteinsconsisting of Hsp70/ATPase or a variety of unrelated proteins (Fas,Siah, Fadd) containing N-terminal LexA DNA-binding domains. TA-BAG-2 andTA-BAG-3 demonstrated positive interactions with LexA-Hsc70/ATPase,resulting in transactivation of a lacZ reporter gene that was under thecontrol of LexA operators (FIG. 11A). No interactions with LexA-Fas(cytosolic domain), LexA-Siah, LexA-Fadd, or LexA were detected (seeFIG. 11A) demonstrating that the BAG-2 and BAG-3 proteins interactspecifically with Hsc70/ATPase. Specific two-hybrid interactions betweenHsc70/ATPase and either BAG-2 or BAG-3 were also observed when BAG-2 andBAG-3 were expressed as LexA DNA-binding domain fusion proteins andHsc70/ATPase was fused with a TA domain (see FIG. 11A; right panel).These results demonstrate that similarly to BAG-1, BAG-2 and BAG-3specifically interact with Hsc70/ATPase.

In order to determine whether the BAG proteins are capable of formingheterodimers, coexpression of BAG-2 and BAG-3 in the yeast two-hybridassay was also performed. Coexpression of BAG-2 and BAG-3 failed to showinteraction with BAG-1 or a deletion mutant of BAG-1 (ΔC) which ismissing part of its C-terminal domain required for Hsp70/Hsc70 bindingsuggest that these proteins do not form heterdimers.

D. Isolation and Characterization of the Complete Open Reading FrameSequences of BAG-2 and BAG-3

In order to deduce the complete ORFs of BAG-2 and BAG-3, a λ-phage cDNAlibrary was screened as follows, using hybridization probes derived fromthe two-hybrid screening. A human jurkat T-cell λ-ZapII library cDNAlibrary (Stratagene) was screened by hybridization using ³²P-labeledpurified insert DNA from the longest of the human BAG-2 (clone #11) andhuman BAG-3 (clone #28) cDNA clones. From about one million clonesscreened, 38 BAG-2 and 23 BAG-3 clones were identified, cloned, andtheir cDNA inserts recovered as pSKII plasmids using a helper phagemethod (Stratagene). DNA sequencing of λ-phage derived human BAG-2 cDNAclones revealed an ORF encoding a predicted 211 amino acid protein,preceded by an in-frame stop codon. The longest human BAG-3 λ-phage cDNAclone contains a continuous ORF of 682 amino acids followed by a stopcodon, but without an identifiable start codon (see FIG. 10A).

Although BAG-1L (SEQ ID NO:2), BAG-1 (beginning at residue 116 of SEQ IDNO:2), BAG-2 (SEQ ID NO:4), and BAG-3 (SEQ ID NO:6) all contain ahomologous BAG domain near their C-terminus, the N-terminal regions ofthese proteins are dissimilar. Using a combination of search tools(Prosite Search: PP search, using the Prosite pattern database, BCMSearch Launcher, Baylor College of Medicine, and Blocks Search), it wasdetermined that the BAG-2 N-terminal region contains potential kinasephosphorylation sites but otherwise shares no apparent similarity withother proteins or known functional domains.

In contrast, the predicted N-terminal region BAG-3 contains a WW domainas shown in FIG. 10A. WW domains have been identified in a wide varietyof signaling proteins, including a Yes kinase adaptor protein (YAP), theNa⁺-channel regulator Nedd4, formin-binding proteins, dystrophin, andthe peptidyl prolyl cis-trans-isomerase Pin-1. These roughly 40 aminoacid domains mediate protein interactions and bind the preferred peptideligand sequence xPPxY (Sudol., TIBS, 21: 161-163, 1996, which isincorporated herein by reference).

Example II In Vitro Association of BAG Proteins and Hsc70/ATPase

This example demonstrates that BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ IDNO:6) bind Hsc70/ATPase in various in vitro assays.

A. Solution Binding Assay of BAG-2 and BAG-3 to Hsc70/ATPase

Association of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) withHsc70/ATPase was determine by an in vitro protein binding assay whereHsc70/ATPase or BAG-family proteins were expressed in bacteria asGlutathione S-Transferase (GST) fusion proteins. Purified cDNA sequencesencoding residues 5 to 211 of human BAG-2 (clone #11) and the C-terminal135 amino acids of human BAG-3 (clone #28) (see FIG. 10A) were subclonedinto the EcoRI/Xho I sites of pGEX4T-1 prokaryotic expression plasmid(Pharmacia; Piscataway, N.J.). These plasmids as well as pGEX4T-1-BAG-1,pGEX-4T-1-BAG-1 (ΔC), and pGEX-4T-1-XL which have been describedpreviously (Takayama et al., supra (1997); Xie et al., Biochemistry,37:6410-6418, (1998), which are incorporated herein by reference), wereexpressed in XL-1 blue strain E. Coli (Stratagene, Inc., La Jolla,Calif.). Briefly, a single colony was inoculated into 1 L of LB mediacontaining 50 μg/ml ampicillin and grown at 37° C. overnight. Theculture was then diluted by half with fresh LB/ampicillin and cooled toroom temperature for 1 hr, before inducing with 0.4 mM IPTG for 6 h at25° C.

Cells were recovered and incubated with 0.5 mg/ml lysozyme in 50 mM Tris(pH 8.0), 150 mM NaCl, 1% TWEEN™-20, 0.1% 2-mercaptoethanol, 5 mM EDTA,1 mM PMSF and a mixture of other protease inhibitors obtained fromBoehringer Mannheim (1697498) at room temperature for 0.5 h, followed bysonication. Cellular debris were pelleted by centrifugation at 27,500 gfor 10 min and the resulting supernatants were incubated with 30 ml ofglutathionine-SEPHAROSE™ (Pharmacia) at 4° C. overnight. The resin wasthen washed with 20 mM Tris (pH 8.0), 150 mM NaCl, 0.1% TWEEN™-20, and0.1% 2-Mercaptoethanol until the OD 280 nm reached <0.01. For removal ofGST, the resin with immobilized GST-fusion protein was incubated with10U of thrombin (Boehringer, Inc.) at 4° C. in 20 mM Tris (pH 8.0), 150mM NaCl, 0.1% TWEEN™-20, 0.1% 2-Mercaptoethanol, and 2.5 mM CaCl₂overnight. Released proteins were then purified on MONO Q™ (HR10/10,Pharmacia) by FPLC using a linear gradient of 0.5M NaCl at pH 8.0 anddialyzed into chaperone assay buffer.

The ability of BAG-2 (SEQ ID NO:4) or BAG-3 (SEQ ID NO:6) to bindHsc70/ATPase in solution was then examined. GST control or GST-BAGproteins were immobilized on glutathione-SEPHAROSE™ and tested forbinding to ³⁵S-labeled in vitro translated (IVT) proteins.Immunoprecipitation and in vitro GST-protein binding assays wereperformed as described by Takayama et al., supra (1997), using pCI-NeoFLAG™ or pcDNA3-HA into which human Bag-2 (clone #11) or human BAG-3(clone #28) had been subcloned for in vitro translation of³⁵S-L-methionine labeled proteins or expression in 293T cells. As shownin FIG. 11B, ³⁵S-Hsc70/ATPase bound in vitro to GST-BAG-1, GST-BAG-2,and GST-BAG-3 but not to GST-BAG-1 (.DELTA.C) or several other controlproteins. BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ IDNO:4), and BAG-3 (SEQ ID NO:6) also exhibited little or no binding tothemselves or to each other, demonstrating that these proteins do notstrongly homo- or hetero-dimerize or oligomerize. It should be noted,however, that BAG-2 (SEQ ID NO:4) displayed weak interactions withitself in binding assays and produced a positive result in yeasttwo-hybrid experiments, demonstrating that it can have the ability toself-associate.

B. Binding of BAG Proteins to Hsc70 In Vivo

The ability of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) proteins tointeract in cells with Hsc70 was tested by expressing these proteinswith N-terminal FLAG™ epitope tags in 293T human epithelial cells usingco-immunoprecipitation assays as described previously (Takayama et al.,supra (1997)). cDNAs encoding the λ-phage cloned regions of BAG-2 andBAG-3 were subcloned in-frame into pcDNA3FLAG™. Anti-FLAG™ immunecomplexes prepared from 293T cells after transfection with plasmidsencoding FLAG™-BAG-1, FLAG™-BAG-2, or FLAG™-BAG-3 were analyzed bySDS-PAGE/immunoblot assay. As shown in FIG. 10C, antiserum specific toHsc70 detected the presence of BAG proteins associated with Hsc70,whereas control immune-complexes prepared with IgG 1 as well asanti-FLAG™ immune complexes prepared from cells transfected withFLAG™-tagged control proteins, Daxx and Apaf-1, did not contain Hsc70associated protein. These results further demonstrate that BAG-familyproteins specifically bind to Hsc70. C. BIACORE™ Assay of BAG ProteinBinding to the ATPase Domain of Hsc70

BAG-1 (beginning at residue 116 of SEQ ID NO:2) is known to bind tightlyto the ATPase domain of Hsc70 (Stuart et al., J. Biol. Chem., In Press(1998)). BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) proteins weretherefore, examined for their ability to bind to Hsc70/ATPase. Theaffinity and binding kinetics of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ IDNO:6) to Hsc70 /ATPase was also compared to that of BAG-1 (beginning atresidue 116 of SEQ ID NO:2) for Hsc70 /ATPase, using a surface plasmonresonance technique (BIACORE™) which has been described previously(Stuart et al., supra, (1998) which is incorporated herein byreference).

BAG-family proteins were produced in bacteria and purified to nearhomogeneity as shown in FIG. 12A and described above in Example I. Thepurified BAG-1 (beginning at residue 116 of SEQ ID NO:2), -2 (SEQ IDNO:4), and -3 (SEQ ID NO:6) proteins were then immobilized on biosensorchips and tested for their interactions with Hsc70 in the soluble phase.Kinetic measurements were performed using a BIACORE™-II instrument withCM5 sensor chip and Amine Coupling Kit (Pharmacia Biosensor AB, Sweden).Briefly, for immobilization of proteins, the sensor chip wasequilibrated with HK buffer (10 mM Hepes (pH 7.4), 150 mM KCL) at 5μl/min, then activated by injecting 17 μl of 0.2MN-ethyl-N′-(3-diethylaminopropyl)-carbodiimide and 0.05MN-hydroxysuccinimide (NHS/EDC) followed by 35 μl of the protein ofinterest, in 10mM acetate, pH 3.5-4.5. Excess NHS-ester on the surfacewas deactivated with 17 μl 1M ethanolamine-HCL (pH8.5). Afterimmobilization, 5 μl of regeneration buffer (50 mM phosphate (pH 6.8)and 4M GuHCl) was injected. For binding assays, Hsp70 (Sigma, H8778) wasdissolved in HK buffer, and injected at 10 μl/ml across the preparedsurface at various concentrations. The surface was regenerated aftereach injection with 5 μl of regeneration buffer. The rate constantsκ_(ass) and κ_(diss) were generated with BIAevaluation software 3.01(Pharmacia Biosensor AB). Addition of Hsc70 to chips containing BAG-1(beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4) or BAG-3(SEQ ID NO:6) resulted in concentration-dependent binding, as reflectedby an increase in the Response Units (RU) measured at the chip surface(shown in FIG. 3B). In contrast, Hsc70 failed to display interactions inBIACORE™ assays with a variety of control proteins as well as a mutantof BAG-1 lacking a C-terminal portion of the BAG domain which isrequired for Hsc70-binding (FIG. 3B). Furthermore, flowing of variouscontrol proteins such as GST, BSA and Bcl-XL over the BAG-1 (beginningat residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ IDNO:6) chips resulted in negligible interaction. These results furtherdemonstrate the specificity with which BAG-family proteins interact withand bind to Hsc70.

The rates of Hsc70 binding to BAG-1 (beginning at residue 116 of SEQ IDNO:2), BAG-2 (SEQ ID NO:4), and BAG-3 (SEQ ID NO:6) proteins weresimilar, following pseudo first-order kinetics with estimatedassociation rate constants (κ_(a)) of 2.1, 2.1 and 2.4×10⁵ M⁻¹ sec⁻¹,respectively. After allowing binding of Hsc70 to immobilized BAG-1(beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3(SEQ ID NO:6) to reach plateau levels, the chaperone was removed fromthe flow solution and the dissociation rate was monitored. BAG-1(beginning at residue 116 at SEQ ID NO:2) and BAG-2 (SEQ ID NO:4)exhibited similar dissociation rates, with relatively slow loss of Hsc70from the chip surface, resulting in estimated dissociation rateconstants (κ_(d)) of 3.0 and 5.0×10⁻⁴ sec⁻¹, respectively (see FIG. 3B).In contrast, Hsc70 dissociated more rapidly from biosensor chipscontaining BAG-3 (see FIG. 3B), yielding an estimated κ_(d) of 1.7×10⁻³sec⁻¹. From the kinetic data, the apparent affinities(κ_(D)=κ_(d)/κ_(a)) were calculated for binding of Hsc70 to BAG-1(beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), andBAG-3 (SEQ ID NO:6) and were estimated to equal about K_(D)=1.4 nM,K_(D)=2.4 nM, and K_(D)=7.4 nM, respectively. These results demonstratethat the interactions of BAG-family proteins with Hsc70 occur withapparent affinities sufficient for physiological relevance.

Example III BAG-family Proteins Inhibit Hsp70/Hsc70-dependent ProteinFolding

This example demonstrates that BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ IDNO:6) proteins inhibit Hsp70/Hsc70-dependent refolding of denaturedproteins similarly to a BAG-1 (beginning at residue 116 of SEQ ID NO:2)protein.

The effects of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) protein onHsp70/Hsc70-dependent protein refolding was determined using in vitroprotein refolding assays similar to those described previously byTakayama et al., supra, 1998; Terada et al., J. Cell Biol.,139:1089-1095, 1997, which are incorporated herein by reference.Briefly, luciferase (20 μM) was denatured in 25 mM Hepes-KOH, pH 7.2, 50mM potassium acetate, 5 mM DTT, 6M guanidine hydrochloride at ^(˜)25° C.for 1 h. Denatured luciferase was diluted 1:40 into 25 mM Hepes-KOH, pH7.2, 50 mM potassium acetate, 5 mM DTT. Hsc70 (1.8 μM), DnaJ (StressGen,Inc.) (0.9 μM), and various purified recombinant proteins as indicatedwere added to refolding buffer (30 mM Hepes-KOH, pH 7.6, 120 mMpotassium acetate, 3 mM magnesium acetate, 2 mM DTT, 2.5 mM ATP) with0.2 volume of diluted denatured luciferase to a final concentration of0.1 μM. Luciferase activity was measured after 1.5 hr incubation at 35°C.

The combination of Hsc70 and DnaJ resulted in ATP-dependent refolding ofchemically denatured firefly luciferase, with function of over half thedenatured enzyme restored in a 90 minute reaction, as monitored by achemiluminescence assay. In contrast, neither Hsc70 nor DnaJ alone wereable to induce substantial refolding of denatured luciferase.Furthermore, little spontaneous restoration of luciferase activity wasobserved with control proteins, BSA, GST or Bcl-XL (see FIG. 4A).

Addition of recombinant purified BAG-1 (beginning at residue 116 of SEQID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6) to the aboveassays in amounts equimolar to Hsc70 (1.8 μM) resulted in strikinginhibition of luciferase refolding. BAG-2 (SEQ ID NO:4) and BAG-3 (SEQID NO:6) displayed somewhat greater inhibitory activity than BAG-1(beginning at residue 116 of SEQ ID NO:2) as shown in FIG. 4A. Incontrast, the BAG-1 (ΔC) protein, which fails to bind Hsc70 as well asseveral other control proteins, had no effect on luciferase refolding.

In an additional refolding assay, described previously by Minami et al.,J. Biol. Chem. 271:19617-24, 1996), purified Hsc70 and human DnaJhomolog Hdj-1 (Hsp 40) were used with additional cofactors provided inreticulocyte lysates (5% v:v) to produce a system capable of refoldingdenatured luciferase. Briefly, additional cofactors included,recombinant Luciferase (Promega: QuantiLum™), that had been heatdenatured at 42° C. for 10 min, 1.8 μM Hsc70 (Sigma; purified frombovine brain), 0.9 μM Hsp40, and various recombinant purified proteins.Luciferase activity was measured (Promega luciferase assay kit) using aluminometer (EG&G Berthold, MicroLumat luminometer, Model #LB96P). Allresults were normalized relative to non-denatured luciferase that hadbeen subjected to the same conditions. Control reactions lacking ATP,Hsc70, or Hsp40 resulted in negligible luciferase refolding.

Various amounts of purified BAG-1 (beginning at residue 116 of SEQ IDNO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6), relative to amountsof Hsc70 were used in the above-described protein refolding assay.Addition of BAG-family proteins resulted in a concentration-dependentinhibition of Hsc70 chaperone activity. Furthermore, the BAG-2 (SEQ IDNO:4) and BAG-3 (SEQ ID NO:6) inhibition of Hsc70 chaperone activity wasdemonstrated to be as potent as that observed for BAG-1 (beginning atresidue 116 of SEQ ID NO:2). In contrast, the BAG-1 (ΔC) mutant as wellas other control proteins did not suppress Hsc70-mediated refolding ofdenatured luciferase. These results indicate that BAG-2 (SEQ ID NO:4)and BAG-3 (SEQ ID NO:6) can inhibit Hsc70/Hsp70 dependent proteinrefolding activity to the same extent as BAG-1 (beginning at residue 116of SEQ ID NO:2).

B. BAG Competes with Hip for Binding to Hsc70.

It is known that BAG-1 competes with Hip for binding to Hsc70, withthese proteins exerting opposite effects on Hsc70-mediated proteinrefolding (Hohfeld, J., and Jentsch, S., Embo J., 16:6209-6216, 1997,which is incorporated herein by reference). In order to determinewhether BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) also compete withHip for binding to Hsc70, refolding assays were performed as describedabove in the presence of Hip protein.

Hip was purified as His₆-protein. The fusion protein was induced frompET28-Hip (V. Prapapanich et al., Mol Cell Biol., 18:944-952, 1998,which is incorporated herein by reference) with 0.1mM IPTG at 25° C. for6 h in BL21 cells. Cells from 1 L of culture were resuspended into 50 mlof 50 mM Phosphate buffer (pH 6.8), 150 mM NaCl, and 1% (v/v) TWEEN™-20and then incubated with 0.5 mg/ml lysozyme at 25° C. for 0.5 h, followedby sonication. After centrifugation at 27,500 g for 10 min, theresulting supernatant was mixed with 15 ml nickel resin (Qiagen, Inc.)at 4° C. for 3 h with 25 mM imidazol. The resin was then washed with 50mM phosphate buffer (pH 6.8), 25 mM imidazol, 150 mM NaCl and 0.1%TWEEN™-20 until the OD280 nm reached a value of <0.01. His₆-Hip proteinwas eluted with 250 mM imidazol in washing buffer (Qiagene, Inc.) andpurified on MONO Q™ (HR10/10 Pharmacia) by FPLC using a linear gradientof 0.5M NaCl at pH 8.0, followed by dialysis in chaperone assay buffer.

In the refolding assay reactions, addition of purified Hip at equimolarconcentrations relative to BAG-1 (beginning at residue 116 of SEQ IDNO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6) (1.8 μM) completelynegated the inhibitory effects of the BAG-family proteins on refoldingof denatured luciferase (see FIG. 4C). These results demonstrate thatthe suppression of Hsc70 chaperone activity by BAG-family proteins isreversible, and that Hip antagonizes the effects of not only BAG-1(beginning at residue 116 of SEQ ID NO:2), but also of BAG-2 (SEQ IDNO:4) and BAG-3 (SEQ ID NO:6).

In summary, these results demonstrate that BAG-family proteins allcontain a conserved BAG domain near their C-terminus that bindsHsc70/Hsp70, and that human BAG-family proteins can bind with highaffinity to the ATPase domain of Hsc70 and inhibit its chaperoneactivity through a Hip-repressable mechanism.

Example IV Expanded Nucleic Acid and Amino Acid Sequences for HumanBAG-3, BAG-4 and BAG-5

Following the procedures disclosed herein, the nucleic acid and aminoacids sequences to human BAG-3, BAG-4 and BAG-5 were further expanded.The expanded sequences for BAG-3, BAG-4 and BAG-5 are shown in FIGS. 15,16 and 17, respectively, with their respective sequence identificationnumbers, “SEQ ID NOs”.

1. A substantially purified nucleic acid molecule encoding the aminoacid sequence of SEQ ID NO:8.
 2. The nucleic acid molecule of claim 1,wherein said nucleic acid molecule has the nucleotide sequence SEQ IDNO:7.
 3. A substantially purified nucleic acid molecule consisting of afragment of at least 35 nucleotides of a nucleotide sequence, or thecomplement thereof, encoding a polypeptide comprising the amino acidsequence of SEQ ID NO:8.
 4. The nucleic acid molecule of claim 3,wherein said fragment is at least 45 nucleotides.
 5. The nucleic acidmolecule of claim 3, wherein said fragment is at least 55 nucleotides.6. A substantially purified nucleic acid molecule consisting of afragment of at least 35 nucleotides of the nucleotide sequence of SEQ IDNO:7, or the complement thereof.
 7. The nucleic acid molecule of claim6, wherein said fragment is at least 45 nucleotides.
 8. The nucleic acidmolecule of claim 6, wherein said fragment is at least 55 nucleotides.